Our work indicates that intrathecal administration of NPY acts in a dose- and NPY receptor-dependent manner to reduce the mechanical and thermal hypersensitivity associated with inflammation or nerve injury. The objective of the present application is to establish NPY as an intrinsic pain modulator, and to identify the mechanisms underlying NPY-mediated inhibition of inflammatory or neuropathic pain. The central hypothesis is that tissue or nerve injury sensitizes the dorsal horn to the pain inhibitory actions of NPY receptor signaling. Our approach involves transgenic, biochemical, and anatomical methods to determine: the anti-allodynic actions of intrinsic NPY (Aim 1); the effect of injury on the efficacy of NPY receptor stimulation (Aim 2); the effect of NPY on the release of substance P (Aim 3A); and the effect of NPY on activity of spinal neurons that express the NPY Y1 receptor (Aim 3B). Our long-term goal is to establish the therapeutic potential of NPY receptor agonists for chronic pain. AIM #1 tests the hypothesis that intrinsic NPY reduces allodynia. To address the contribution of endogenous NPY to chronic pain, Aim #1 will utilize transgenic mice (NPYtet) that contain a doxycycline (Dox)- regulated cassette (tetracycline trans-activator, tTA) at the promotor region of the npy locus. We describe data indicating that inducible NPY depletion increases behavioral signs of tactile and cold allodynia in the spared nerve injury (SNI) model of neuropathic pain. These data lead us to determine the effect of conditional NPY knockdown on the induction, maintenance, and reinstatement of chronic pain. We predict that NPY depletion will speed the development, increase the intensity, and extend the duration of the allodynia induced by nerve injury or adjuvant-induced inflammation. AIM #2 tests the hypothesis that injury increases NPY receptor signaling in the dorsal horn. To determine whether injury increases NPY receptor-G protein coupling, we will evaluate Y1- and Y2-agoniststimulated GTP?S binding after inflammation, nerve injury, or sham surgery in NPYtet mice. Because receptor expression levels must be taken into account when assessing the efficacy of NPY receptor stimulation, we will also measure Y1 and Y2 expression after inflammation, nerve injury, or sham surgery. AIM #3 will test the hypothesis that NPY inhibits spinal pronociceptive neurotransmission. Y1 receptors decorate presynaptic terminals in the dorsal horn. Based on our preliminary results, we predict that intrathecal NPY will dose- and receptor-dependently reduce the injury-induced release of SP (using microdialysis and injury-induced NK1 receptor internalization). Because Y1 receptors also label spinal neurons, we will test for postsynaptic actions of NPY. We will determine the effect of intrathecal NPY on SP-induced nociceptive behavior and NK1 receptor internalization. Our preliminary data with the selective neurotoxin NPY-saporin suggest that Y1-expressing cells in the dorsal horn facilitate pain transmission. We predict that the activity of this population of spinal neurons will be decreased by NPY administration, and increased by NPY knockdown.